Recombinant DNA Technology and Gene Expression
Recombinant DNA technology involves insertion of specific DNA sequences into a DNA vehicle (vector) to form a recombinant DNA molecule which is capable of replication in a host cell. Generally, the inserted DNA sequence is foreign to the recipient DNA vehicle, i.e., the inserted DNA sequence and the DNA vector are derived from organisms which do not exchange genetic information in nature, or the inserted DNA sequence may be wholly or partially synthetically made. Several general methods have been developed which enable construction of recombinant DNA molecules.
Regardless of the method used for construction, the recombinant DNA molecule must be compatible with the host cell, i.e., capable of autonomous replication in the host cell or stably integrated into one or more of the host cell's chromosomes or plasmids. The recombinant DNA molecule should preferably also have a marker function which allows the selection of the desired recombinant DNA molecule(s). In addition, if all of the proper replication, transcription, and translation signals are correctly arranged on the recombinant vector, the foreign gene will be properly expressed in, e.g., the transformed bacterial cells, in the case of bacterial expression plasmids, or in permissive cell lines or hosts infected with a recombinant virus or carrying a recombinant plasmid having the appropriate origin of replication.
Different genetic signals and processing events control levels of gene expression such as DNA transcription and messenger RNA (mRNA) translation. Transcription of DNA is dependent upon the presence of a promoter, which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and furthermore, procaryotic promoters are not recognized and do not function in eucaryotic cells.
Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals, which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno (S/D) sequence (Shine, J. and Dalgarno, L., 1975, Nature 254:34-38) on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal (formyl-) methionine of the protein. The S/D sequences are complementary to the 3' end of the 16S rRNA (ribosomal RNA), and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome (Shine, J. and Dalgarno, L., 1975, Nature 254:34-38).
Successful expression of a cloned gene requires sufficient transcription of DNA, translation of the mRNA, and in some instances, post-translational modification of the protein. Expression vectors have been used to express genes under the control of an active promoter in a suitable host, and to increase protein production.
Vaccines
The development of vaccines for the prevention of viral, bacterial, fungal or parasitic diseases is the focus of much research effort.
Traditional ways of preparing vaccines include the use of inactivated or attenuated pathogens. A suitable inactivation of the pathogenic microorganism renders it harmless as a biological agent but does not destroy its immunogenicity. Injection of these "killed" particles into a host will then elicit an immune response capable of preventing a future infection with a live microorganism. However, a major concern in the use of killed vaccines (using inactivated pathogen) is failure to inactivate all the microorganism particles. Even when this is accomplished, since killed pathogens do not multiply in their host, or for other unknown reasons, the immunity achieved is often incomplete, short lived and requires multiple immunizations. Finally, the inactivation process may alter the microorganism's antigens, rendering them less effective as immunogens.
Attenuation refers to the production of strains of pathogenic microorganisms which have essentially lost their disease-producing ability. One way to accomplish this is to subject the microorganism to unusual growth conditions and/or frequent passage in cell culture. Mutants are then selected which have lost virulence but yet are capable of eliciting an immune response. Attenuated pathogens often make good immunogens as they actually replicate in the host cell and elicit long lasting immunity. However, several problems are encountered with the use of live vaccines, the most worrisome being insufficient attenuation and the risk of reversion to virulence.
An alternative to the above methods is the use of subunit vaccines. This involves immunization only with those components which contain the relevant immunological material.
Vaccines are often formulated and inoculated with various adjuvants. The adjuvants aid in attaining a more durable and higher level of immunity using smaller amounts of antigen or fewer doses than if the immunogen were administered alone. The mechanism of adjuvant action is complex and not completely understood. However, it may involve the stimulation of cytokine production, phagocytosis and other activities of the reticuloendothelial system as well as a delayed release and degradation of the antigen. Examples of adjuvants include Freund's adjuvant (complete or incomplete), Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), the pluronic polyol L-121, Avridine, and mineral gels such as aluminum hydroxide, aluminum phosphate, etc. Freund's adjuvant is no longer used in vaccine formulations for humans because it contains nonmetabolizable mineral oil and is a potential carcinogen.
Live, attenuated Salmonella have been demonstrated to be capable of stimulating a protective immune response against challenge with the homologous, virulent strain (Germanier, R. and Furer, E., 1975, J. Infect Dis. 181:533; Germanier, R., 1984, in Bacterial Vaccines, Academic Press, N.Y., pp. 137-165; Levine, M. M., et al., 1983, Microbiol. Rev. 47:510; Wahdan, M. H., et al., 1982, J. Infect. Dis. 145:292; Hoiseth, S. K. and Stocker, B. A. D., 1981, Nature 291:238; Stocker, B. A. D., et al. 1982, Dev. Biol. Std. 53:47; Lindberg, A. A. and Robertsson, J. A., 1983, Infect. Immun. 41:751; Robertsson, J. A., et al., 1983, Infect. Immun. 41:742; Smith, B. P., et al., 1984, Am. J. Vet. Res. 45:2231; Smith, B. P., et al., 1984, Am. J. Vet. Res. 45:59; Moser, I., et al., 1980, Med. Microbiol. Imm. 168:119). In addition, several investigators have utilized attenuated Salmonella harboring plasmids encoding foreign antigens to deliver these foreign antigens to the immune system (Formal, S. B., et al., 1981, Infect. Immun. 34:746; U.S. Pat. No. 4,632,830 by Formal et al.; Clements, J. D., et al., 1986, Infect. Immun. 53:685; Maskell, D. J., et al., 1987, Microb. Path. 2:211; Brown, A., et al., 1987, J. Infect. Dis. 155:86; Dougan, G., et al., 1987, Parasite Immun. 9:151).
The repeating immunodominant epitope associated with the circumsporozoite protein of Plasmodium species is considered the target for protective humoral (and possible cell-mediated) responses against malaria sporozoites (Miller, L. H., et al., 1986, Science 234:1349); monoclonal antibodies have been described which recognize these molecules and are able to passively protect naive recipient animals. Two vaccines based on these repeating epitopes have been tested in humans, and have been shown to induce a protective immune response in some individuals (Ballou, W. R., et al., 1987, Lancet i:1277; Herrington, D., et al., 1987, Nature 328:257).
Cholera toxin is the prototype of a family of bacterial enterotoxins which mediate diarrheal disease and are related in structure, function and immunogenicity. Other members of this family include the heat-labile toxin of E. coli isolated from humans (Yamamoto, T. and Yokota, T., 1983, J. Bacteriology 155:728) and from pigs (Leong, J., et al., 1985, Infect. Immun. 48:73), and toxins from Salmonella typhimurium (Finkelstein, R. A., et al., 1983, FEMS Microbiology Letters 17:239) and from Campylobacter jejuni (Walker, R. I., et al., 1986, Microbiology Rev. 50:81). Common to all of these toxins is an A subunit which mediates ADP-ribosyltransferase activity, resulting in the activation of adenylate cyclase, ultimately leading to death of the target cell. In addition, all of these toxins contain an immunologically dominant B subunit which mediates binding of the holotoxin to the target cell. The B subunit by itself is non-toxic, and immunization with this molecule induces the formation of toxin-neutralizing antibodies.
Vaccines based on the formation of toxin-neutralizing antibody responses by immunization with the non-toxic binding subunits of bacterial exo-toxins (Cholera toxin, heat-labile toxin of E. coli) have been proposed (Jacob, C. O., et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7611; Jacob, C. O., et al., 1984, EMBO J. 3:2889).
The hepatitis B virion is a 42 nm enveloped structure containing a small DNA genome. The envelope proteins are encoded by the S gene (preS, preS.sub.2 and S), one of the four open reading frames of the HBV genome (Tiollais, P. et al., 1985, Nature 317:489). These polypeptides contain the hepatitis B surface antigen (HBsAg). HBsAg particles derived from human plasma or similar HBsAg particles produced by recombinant DNA methods (some of which lack preS epitopes) have been shown to elicit a protective immune response and the purified particles represent current vaccines for HBV (Krugman, S., 1982, J. Am. Med. Assoc. 247:2012).
Flagellin
Flagella are organelles which are involved in locomotion of bacterial cells. The synthesis of structural proteins and the actual function of assembled flagella is a complex process involving the interactions of many genes and gene products (reviewed by Iino, T., 1977, Ann. Rev. Genet. 11:161). More than thirty-five genes have been defined which play a role in flagellar function in E. coli, and gene products for at least seventeen of these have been identified. The actual flagellar organelle is composed of three major structural elements, and spans from the cell cytoplasm, across the cell membranes, and culminates in a large extracellular domain. The filament is composed of a single subunit protein, flagellin, and is the major structural component of the organelle, accounting for more than 95% of the total mass. The structural genes for flagellin have been termed H1 and H2 in Salmonella (Iino, T., 1969, Bacteriol. Rev. 33:454-475), H in Bacillus subtilis (Joys, T. M. and Frankel, R. W., 1967, J. Bacteriol. 94:32-37) and Pseudomonas aeruginosa (Iino, T., 1969, Ann. Rep. Natl. Inst. Genet. Jpn. 20:94), and hag in E. coli (Armstrong, J. B. and Adler, J., 1969, J. Bacteriol. 97:156-161). The basal body is the most complex part of the organelle and serves both to anchor the organelle to the cell and as part of the motor-like apparatus which rotates the filament. Finally, the hook serves to attach the filament to the basal body.
Rotation of the filament is responsible for flagella-mediated locomotion. Each filament consists of several thousand copies of the flagellin subunit resulting in a helical structure typically 5-10 u in length (for most E. coli and Salmonella species; MacNab, P., 1987, in Eschericia coli and Salmonella typhimurium, Neidhardt, F. C., Eds. American Society for Microbiology, Washington, D.C., pp. 70-83). Mutations in the flagellin structural gene have been observed to produce changes in efficiency of filament formation, filament shape, sensitivity to flagellotropic phage, and/or the antigenic specificity of the flagella (Yamaguchi, S. and Iino, T., 1969, J. Gen. Microbiol. 55:59-74; Iino, T., et al., 1974, J. Gen. Microbiol. 81:37-45;
Horiguchi, T., et al., 1975, J. Gen. Microbiol. 91:139-149). Filaments are assembled extracellularly by sequential addition of flagellin monomers to the distal end of the growing filament, and the rate of elongation decreases inversely with the length of the filament until growth ceases, thus regulating filament length.
Flagella are found primarily, although not exclusively, on the surface of rod and spiral shaped bacteria, including members of the genera Escherichia, Salmonella, Proteus, Pseudomonas, Bacillus, Campylobacter, Vibrio, Treponema, Legionella, Clostridia, Caulobacter, and others. These flagella, although they perform the same function, are polymorphic in molecular weight across genera, ranging from 28-66 kd. A high degree of antigenic polymorphism is seen even within a single genus, such as Salmonella, and is useful for identifying individual serotypes within a single species (Edwards, P. R. and Ewing, W. H., 1972, Identification of Enterobacteriaceae, 3d ed., Burgess Publishing Co., Minneapolis, Minn.). Structural analyses of several bacterial flagella have revealed a common architecture among filaments isolated from different bacteria (Wei, L.-N. and Joys, T. M., 1985, J. Mol. Bio. 186:791; DeLange, R. J., et al., 1976, J. Biol. Chem. 251:705; Gill, P. R. and Agabian, J., Biol. Chem. 258:7395). Most striking is a high degree of protein sequence homology at the amino and carboxy termini of these molecules, and the presence of a polymorphic central region which is responsible for the antigenic diversity among different flagella.
Host immune responses to antigens on the surface of bacteria have been well documented (Horowitz, S. A. et al., 1987, Infect. Immun. 55:1314; Naito, Y., et al., 1987, Infect. Immun. 55:832; Zhang, Y. X., et al., 1987, J. Immunol. 138:575; Norgard, M. V., 1986, Infect. Immun. 54:500; Nagy, L. K., 1985, Vet. Rec. 117:408; Levine, M. M., et al., 1984, Infect. Immun. 44:409; Zak, K., et al., 1984, J. Infect. Dis. 149:166). Flagella, and especially flagellar filaments, have been shown to be potent immunogens under conditions of natural infection and artificial immunization, and in some cases, the response to antigenic determinants present on flagella have been shown to be protective (Young, R. J., et al., 1979, Infect. Immun. 25:220; Eubanks, E. R., et al., 1976, Infect. Immun. 15:533; Smith, H. L., Jr., 1974, App. Micro. 27:375; Dwyer, J. M. and Mackay, I. R., 1972, Int. Arch. Allergy Appl. Imm. 43:434; Ebersole, J. L. and Molinari, J. A., 1976, Infect. Immun. 13:53; Ebersole, J. L., et al., 1975, Infect. Immun. 12:353; Stevenson, J. R. and Stronger, K. A., 1980, Am. J. Vet. Res. 41:650; Tamura, Y., et al., 1984, Micro. Imm. 28:1325). Kuwajima (1988, J. Bact. 170:485) has described the production of E. coli mutants with altered flagella antigenicity by the introduction of deletions into the central region of the flagellin hag gene.
U.S. Pat. No. 4,702,911 discloses the use of purified subunits of bacterial pili, hairlike organelles attached to the outer bacterial surface, in vaccine formulations.
International PCT Publication No. WO 87/02385, published Apr. 23, 1987, discloses the expression of a proinsulin sequence and a beta-lactamase sequence as fusion proteins with the B. subtilis flagellin hag gene.